Project Summary/Abstract Mitogen activated protein kinase (MAPK)-targeted therapy (MTT) currently is approved for treating metastatic melanoma patients with tumors harboring an oncogenic mutation in BRAF. Anti-PD-1 antibodies are the standard front-line approach for patients with metastatic melanoma, regardless of genotype, but responses only are seen in 40-45% of patients. To improve outcomes with MTT and anti-PD1, more effective approaches are needed. We recently demonstrated that MTT leads to increased tumor infiltrating lymphocyte (TIL) number, clonality, and effector function, as well as increased immune exhaustion markers such as PD-1, its ligand PD- L1, and TIM3. We hypothesize that these MTT-associated tumor microenvironment changes enhance immune responses in the tumor microenvironment and have the potential to convert an immunologically non-responsive tumor microenvironment into one more responsive to subsequent anti-PD1 therapy. To test our hypothesis, we have opened a trial (NCT031429029) that incorporates a lead-in phase of MTT, a brief period of concomitant MTT and anti-PD-1 therapy with pembrolizumab (pembro), followed by single-agent pembro (Aim 1). We will investigate the clinical benefit rate (CBR) of abbreviated MTT in combination with pembro and determine if MTT-associated effects on the tumor-immune microenvironment are associated with improved CBR at 24 weeks. As part of the trial, serial biopsies (pretreatment, post-MTT lead-in, and on-MTT plus pembro) and peripheral blood will be collected, and our team of clinical, translational, and basic investigators will use these samples to identify biomarkers associated with response and to develop a mechanistic understanding of the effects of MTT alone and in combination with anti-PD-1 antibody therapy on the tumor microenvironment. We also will evaluate the effects of anti-PD-1 and MTT on T cell responses in a GEMM melanoma model and in patients (Aim 2). We will analyze T cell subsets in the mouse melanoma model, assessing the generation of memory T cells in mice that control tumors using tumor rechallenge studies. We will characterize T cell responses in the patients enrolled in the clinical trial in Aim 1, focusing on features of effector, memory, and exhausted T cell subsets. Lastly, we will examine memory populations in melanoma patients who are long- term survivors after treatment with immune checkpoint inhibitors or MTT. Finally, we will pair these translational studies with a complementary mouse melanoma model to determine how this therapy affects the generation, function and maintenance of T cell subsets, and identify new potential therapeutic strategies using in vivo CRISPR screens of mouse melanoma cells. (Aim 3)